The long-term goal of this laboratory continues to be the characterization of N-acetylaspartylglutamate (NAAG) as a major peptide co-neurotransmitter and group II metabotropic glutamate receptor agonist. NAAG peptidase activities regulate peptide levels following synaptic release. Inhibition of NAAG peptidase activities has therapeutic efficacy in animal models of inflammatory pain, allodynia, stroke, diabetic neuropathy, ALS, and schizophrenia. The immediate aims are to contrast the cellular expression of two NAAG peptidases that we have cloned, GCPII and III, and to analyze the consequences of inhibiting their activities. Among the hypotheses to be tested are: 1) GCPII and GCPIII function in distinct cell types and locations to regulate NAAG levels following synaptic release; 2) GCPII knock out mice are dependent upon GCPIII activity to retain normal neurological function; 3) increasing extracellular NAAG levels via peptidase inhibition in the brain and/or at the spinal-sensory level influences inflammatory and neuropathic pain perception via group II mGluRs; 4) NAAG acting via group II mGluRs decreases Substance P and CGRP release and EPSCs at sensory spinal synapses; 5) NAAG peptidase inhibition acts centrally to suppress pain-induced glial activation and peripherally to reduce PGE2-induced hyperalgesia and potentiation of capsaicin responses; 6) mGluRS receptors are negatively coupled to cGMP as well as cAMP; 7) activation of glial group II mGluRs by inhibition of NAAG peptidase increases expression of glutamate transporters, GLT-1 and GLAST. The effect of inhibition of GCPIII on total NAAG peptidase activity will be tested in vivo and in vitro using GCPII knock out mice. The influence of peptidase inhibition on inflammatory and neuropathic pain perception will be determined in the rat model and in wild type, heterozygous and GCPII knock out mice. The influence of elevated NAAG levels in vivo, obtained by inhibition of NAAG peptidase activity, will be tested in rat brain and spinal cord and in transgenic mice expressing GLT-1-GFP and GLAST-GFP fusion proteins.